Mosquito-borne diseases are the number-one killers of humans worldwide. A major obstacle in controlling these diseases is that mosquitoes have developed resistance to insecticides. Our long-term goals are to determine the roles of genes that control the mosquito's response to insecticides and to use this information to develop novel approaches to control mosquito vectors. A large number of studies has shown that multiple, complex mechanisms or genes may be responsible for insecticide resistance. However, many questions remain, including these 2 that we seek to answer: 1) how many detoxification gene families and individual genes are involved in insecticide resistance in a single organism, such as the mosquito? And 2) are there genes, in addition to metabolic detoxification genes, the up-regulation of which confers resistance? The Liu laboratory has recently identified 22 novel genes that are over-expressed in Culex HAmCq mosquitoes. These genes are known to be vital for cellular and molecular metabolism, signal transduction and regulation, vesicular and molecular transport, and protein biosynthesis and ubiquitination, but most of them have not been implicated in insecticide resistance. The objective of the proposed project is to determine the potential roles of the 22 up-regulated genes in insecticide resistance in mosquitoes. Our hypothesis is that among the 22 up-regulated genes in our study, the genes involved in insecticide resistance (the key genes) exhibit increased expression, increased protein activities, and increased protein expression following insecticide selection. Our specific aims are to: 1) Define the relationship between the levels of resistance and the levels of gene expression, enzyme activity, and protein expression in each of the 22 up-regulated genes in the susceptible S-Lab strain;the field permethrin-resistant strains of HAmCqG0, MAmCqG0, and BAmCqG0;8 generations of permethrin-selected HAmCqG0 offspring (HAmCqG1 to HAmCqG8);and the 8th generation of the permethrin-selected S-Lab strain (S-Lab-PerG8) and 2) Determine the relationship between the silenced expression levels of key genes in HAmCqG8 and their corresponding levels of insecticide resistance. Characterizing roles of the 22 up-regulated genes in insecticide resistance is a necessary and fundamental first step in determining resistance mechanisms, understanding the function of these genes in mosquito insecticide resistance, and ultimately providing important new information that can be used for basic studies of resistance-gene interaction and regulation, and for applied studies in developing novel approaches for controlling mosquitoes. PUBLIC HEALTH RELEVANCE The proposed project seeks to identify the role of up-regulated genes in insecticide resistance in mosquitoes by defining the relationship between the levels of resistance and the levels of gene expression, enzyme activity, and protein expression of the up-regulated genes in the following strains: the susceptible S-Lab strain, the field permethrin resistant strains (HAmCqG0, MAmCqG0, and BAmCqG0), 8 generations of permethrin-selected HAmCqG0 offspring (HAmCqG1 to HAmCqG8);and the 8th generation of permethrin-selected S-Lab strain (S-Lab-PerG8). We will silence (reduce) the expression of key genes in HAmCqG8 to determine their functions in insecticide resistance.